Affinity peptides toward infliximab

ABSTRACT

We have disclosed affinity peptides toward infliximab. More specifically we have disclosed an affinity biomatrix where the affinity peptide is covalently attached to a biocompatible, biodegradable polymer. The affinity biomatrix is useful in preparing controlled release devices for infliximab.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 27, 2012, isnamed RTX5041U.txt and is 10,394 bytes in size.

FIELD OF THE INVENTION

The invention relates to specific affinity peptides toward infliximab.In addition, the invention relates the use of these affinity peptides incontrolled release devices for infliximab.

BACKGROUND OF THE INVENTION

Infliximab is a chimeric IgG1κ monoclonal antibody, which is a type ofprotein that recognizes, attaches to, and blocks the action of tumornecrosis factor-alpha (TNF-alpha). Infliximab is currently sold underthe tradename REMICADE by Centocor Ortho Biotech, Inc., in Horsham, Pa.Infliximab has been used for the treatment of inflammatory disorders,such as plaque psoriasis, rheumatoid arthritis, psoriatic arthritis,adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis,and ankylosing spondylitis. Currently, infliximab is given by IVinfusion and it's half-life is approximately eight weeks. Initially,patients receive three infusions (5 mg/kg) given at 0, 2 and 6 weeks.Following, the patients are dosed (5 mg/kg) every eight weeks. Due tofrequent dosing and patient visits to the clinic, the cost associatedwith infliximab is high.

Therefore, there is a need for a controlled release device forinfliximab to eliminate the frequent dosing and doctor visits for thepatient and provide a cost effective treatment. Standard methods forpreparing controlled release devices include the use of polymericmatrices, typically in the form of microspheres, rods, sheets orpellets, which are used to encapsulate the active agent. A variety oftechniques are known by which active agents can be incorporated intopolymer matrices. Examples include solvent evaporation, spray drying,emulsification, melt blending and simple physical mixing of particles ofdiscrete size or shape. None of these approaches may be applied toincorporate peptides or proteins into the polymers due to the delicatenature of these molecules. Peptides and proteins are susceptible todenaturation by solvents, by emulsification, by heat and, in particular,by terminal sterilization.

Therefore, there is a need for a method of making a controlled releasedevice for infliximab, where the method does not denature or otherwiseinactivate the activity of the protein. A controlled release device forinfliximab is also desired which provides a localized, sustained releaseof the protein, eliminates the need for frequent dosing and doctor'svisits for the patient, and provides a cost effective treatment.

SUMMARY OF THE INVENTION

We have described herein specific affinity peptides toward infliximab.These affinity peptides, which have a specific affinity for infliximab,are useful in preparing an affinity biomatrix. Controlled releasedevices for infliximab are also described which are prepared from theaffinity biomatrix and infliximab.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Amino acid sequences of the infliximab affinity peptides,containing linear and disulfide-constrained peptides.

FIG. 2A. Pictorial representation of a controlled release devicecomprising an affinity biomatrix and infliximab.

FIG. 2B. Pictorial representation of the surface of a controlled releasedevice comprising an affinity biomatrix and infliximab.

FIG. 3. Infliximab phage ELISA assay confirming that the phage-displayedpeptides generated from the phage selection bind to infliximab.

FIG. 4A. Cross-reactivity phage ELISA assays toward mAb competitor.

FIG. 4B. Cross-reactivity phage ELISA assays toward human IgG.

FIG. 4C. Cross-reactivity phage ELISA assays toward human serum albumin(HSA).

FIG. 4D. Cross-reactivity phage ELISA assays toward collagen.

FIG. 5. Concentration dependent affinity peptide binding to infliximabdetermined using ELISA.

FIG. 6A. Amino acid sequences (SEQ ID NO: 24 and SEQ ID NO: 25) of thealkyne peptides used for the conjugation of SEQ ID NO: 2 and SEQ ID NO:10 respectively, to the azide-modified collagen matrix using clickchemistry.

FIG. 6B. Amino acid sequences (SEQ ID NO: 26 and SEQ ID NO: 27) of thelysine terminated peptides used for the conjugation of SEQ ID NO: 2 andSEQ ID NO: 10 respectively, to the aldehyde functionalized hyaluronicacid matrix using schiff base chemistry.

FIG. 7. Functional interference of affinity peptides on infliximabTNF-alpha binding by assessment of viability of mouse WEHI cells after24 hour incubation in the presence of infliximab.

FIG. 8A. In vitro release study, performed in phosphate buffered saline(PBS) containing 2% heat inactivated fetal bovine serum (FBS), showingthe amount of infliximab released from the collagen biomatrix andcollagen affinity biomatrix discs over a time period of 34 days.(Initial loading concentration=200 micrograms)

FIG. 8B. In vitro release study, performed in phosphate buffered saline(PBS) containing 2% heat inactivated fetal bovine serum (FBS), showingthe amount of infliximab released from the collagen biomatrix andcollagen affinity biomatrix discs over a time period of 34 days.(Initial loading concentration=50 micrograms)

FIG. 9. Mutant phage ELISA showing linear fragments of SEQ ID NO: 18that bind to infliximab.

FIG. 10. Amino acid sequences of the linear fragments of SEQ ID NO: 18that bind to infliximab.

DETAILED DESCRIPTION OF THE INVENTION

We describe herein, affinity peptides toward infliximab. In oneembodiment, the infliximab is an anti-inflammatory, chimeric IgG1κmonoclonal antibody which blocks TNF-alpha receptor binding. Affinitypeptide toward infliximab indicates that the peptide has a specificbinding affinity toward infliximab. The peptide may be either linear ordisulfide constrained. In one embodiment, the affinity peptide towardinfliximab is any one of the affinity peptides shown in FIG. 1. Inanother embodiment, the affinity peptide may be a fragment of any one ofthe affinity peptides shown in FIG. 1. While the affinity peptide hasspecific affinity toward infliximab, the affinity peptide does notinterfere with the binding of TNF-alpha to infliximab. These affinitypeptides are useful in the preparation of controlled release devices forinfliximab.

The affinity peptides toward infliximab shown in FIG. 1 were identifiedusing a pIX phage display technology described in U.S. Pat. No.6,472,147. Primary peptide phage libraries with high complexity (10⁹peptides sequences per library) were used to select for affinitypeptides toward infliximab. More specifically, biotinylated-infliximabwas immobilized on a streptavidin-coated ELISA plate and phage whichdisplay the peptide sequences on the pIX minor coat protein wereintroduced. The phage that did not bind to infliximab were washed awayand the phage that did bind to infliximab were isolated and amplified.The isolated phage that bound to infliximab were used as input for thesecond round of the selection following the procedure stated above for atotal of three rounds. At the end of the third round the phage thatbound to infliximab were sequenced to determine the peptide which isresponsible for binding to infliximab. Peptides having affinity towardinfliximab are listed in FIG. 1.

The affinity peptides toward infliximab are useful in preparing anaffinity biomatrix. The affinity biomatrix is prepared by covalentlyattaching at least one affinity peptide toward infliximab, as describedabove, to a biocompatible, biodegradable polymer. The affinity biomatrixmay be prepared such that the affinity peptide toward infliximab ispresent in the bulk of the polymer, on the surface of the polymer, orboth in the bulk of the polymer and on the surface of the polymer.Furthermore, the affinity biomatrix is useful as a controlled releasedevice for infliximab. Representative embodiments of controlled releasedevices are shown pictorially in FIG. 2A and FIG. 2B. The controlledrelease device for infliximab comprises an affinity biomatrix andinfliximab.

The biocompatible, biodegradable polymer used to prepare the affinitybiomatrix may be natural polymers, synthetic polymers, and combinationsthereof. The biodegradable polymers readily break down into smallsegments when exposed to moist body tissue. The segments then either areabsorbed by the body, or passed by the body. More particularly, thebiodegraded segments do not elicit permanent chronic foreign bodyreaction, because they are absorbed by the body or passed from the body,such that no permanent trace or residual of the segment is retained bythe body.

Suitable natural polymers include, but are not limited to proteins suchas, collagen, elastin, keratin, silk, glucosaminoglycans (GAGs),thrombin, fibronectin, gelatin, fibrin, tropoelastin, polypeptides,laminin, proteoglycans, fibrin glue, fibrin clot, platelet rich plasma(PRP) clot, platelet poor plasma (PPP) clot, self-assembling peptidehydrogels, and atelocollagen; polysaccharides such as, starch, pectin,cellulose, alkyl cellulose (e.g. methylcellulose), alkylhydroxyalkylcellulose (e.g. ethylhydroxyethyl cellulose), hydroxyalkyl cellulose(e.g. hydroxylethyl cellulose), cellulose sulfate, salts ofcarboxymethyl cellulose, carboxymethyl cellulose, carboxyethylcellulose, chitin, carboxymethyl chitin, hyaluronic acid, salts ofhyaluronic acid, alginate, cross-linked alginate alginic acid, propyleneglycol alginate, glycogen, dextran, dextran sulfate, curdlan, pectin,pullulan, xanthan, chondroitin, chondroitin sulfates, carboxymethyldextran, carboxymethyl chitosan, chitosan, heparin, heparin sulfate,heparan, heparan sulfate, dermatan sulfate, keratan sulfate,carrageenans, chitosan, starch, amylose, amylopectin,poly-N-glucosamine, polymannuronic acid, polyglucuronic acidpolyglucuronic acid), and derivatives; polynucleotides such as,ribonucleic acids, deoxyribonucleic acids, and combinations thereof.

In one embodiment the natural polymer is collagen. In yet anotherembodiment the natural polymer may be obtained from decellularizedtissue. The decellularized tissue may be obtained from autogeneictissue, allogeneic tissue or xenogeneic tissue. Suitable decellularizedtissues include, but are not limited to skin, omentum, periosteum,perichondrium, synovium, fascia, mesenter, bone, sinew, and the like. Inanother embodiment, the natural polymer is a polysaccharide. In yetanother embodiment, the polysaccharide is hyaluronic acid.

Examples of suitable synthetic polymers include, but are not limited toaliphatic polyesters, poly(amino acids), copoly(ether-esters),polyalkylene oxalates, polyamides, poly(iminocarbonates),polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesterscontaining amine groups, poly(anhydrides), polyphosphazenes,biomolecules and blends thereof. For the purpose of this inventionaliphatic polyesters include, but are not limited to homopolymers andcopolymers of lactide (which includes lactic acid, d-, l- and mesolactide), glycolide (including glycolic acid), epsilon-caprolactone,p-dioxanone (1,4-dioxan-2-one), trimethylene carbonate(1,3-dioxan-2-one), alkyl derivatives of trimethylene carbonate,delta-valerolactone, beta-butyrolactone, gamma-butyrolactone,epsilon-decalactone, hydroxybutyrate (repeating units), hydroxyvalerate(repeating units), 1,4-dioxepan-2-one (including its dimer1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one,6,6-dimethyl-1,4-dioxan-2-one 2,5-diketomorpholine, pivalolactone,alpha, alpha-diethylpropiolactone, ethylene carbonate, ethylene oxalate,3-methyl-1,4-dioxane-2,5-dione, 3,3-diethyl-1,4-dioxan-2,5-dione,6,8-dioxabicycloctane-7-one and polymer blends thereof.

Suitable aliphatic polyesters include, but are not limited tohomopolymers and copolymers of lactide (which includes lactic acid,D-,L- and meso lactide), glycolide (including glycolic acid),epsilon-caprolactone, p-dioxanone (1,4-dioxan-2-one), trimethylenecarbonate (1,3-dioxan-2-one), alkyl derivatives of trimethylenecarbonate, delta-valerolactone, beta-butyrolactone, gamma-butyrolactone,epsilon-decalactone, hydroxybutyrate (repeating units), hydroxyvalerate(repeating units), 1,4-dioxepan-2-one (including its dimer1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one,6,6-dimethyl-1,4-dioxan-2-one and polymer blends thereof.

In one embodiment, the biocompatible, biodegradable polymer and theaffinity peptides toward infliximab have suitable reactive groups andcorresponding functional groups to covalently attach the affinitypeptide to the polymer. The reactive groups and functional groups may beon the polymer, the affinity peptide, and combinations thereof. Suitablereactive groups include, but are not limited to aryl azide,carbodiimide, hydrazine, hydroxymethyl phosphine, imidoester,isocyanate, carbonyl, maleimide, NHS-ester, PFP-ester, thiol, pyridyldisulfide and/or vinyl sulfone and the like. Suitable functional groupsinclude, but are not limited to hydroxyl, carboxyl, aldehyde, ester,thiol, amine, alkene, alkyne, alkyl halide, hydrazine and/or azidefunctional groups. One of skill in the art of organic chemistry orpolymer chemistry would be able to functionalize the affinity peptideand the polymer with the above suitable reactive groups andcorresponding functional groups to covalently attach the affinitypeptide to the polymer. For example, in the case of collagen the aminefunctional groups on the lysine residues displayed from collagen can becoupled to the C-terminal carboxyl group of an affinity peptide usingsimple carbodiimide chemistry. Alternatively, in the case of hyaluronicacid the amine functional groups on the lysine residues displayed fromthe affinity peptide can be coupled to hyaluronic acid by using simpleoxidation chemistry.

Optionally, a linker sequence may be added to the peptides shown in FIG.1 in order to increase the availability of the affinity peptide to bindto infliximab. The linker sequence can be (SGG)_(n) or (XXX)_(n), whereX may be any combination of serine, glycine, alanine or threonine. Inone embodiment, the number of repeat units n is from about 1 to about10. In another embodiment, the number of repeat units n is from about 3to about 5.

For example, the affinity biomatrix may be prepared from soluble and/orfibrillar Type I bovine collagen. Briefly, a homogenized collagensuspension in water, having a concentration of from about 10 mg/mL toabout 100 mg/mL, is cast into a mold and then lyophilized. After athermal dehydration step, the affinity peptides are conjugated to thecollagen using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC)/N-hydroxysulfosuccinimide (Sulfo-NHS) couplingchemistry. Alternatively, after a thermal dehydration step andfunctionalization of the collagen with an azide, alkyne-modifiedaffinity peptides are conjugated to the collagen with a copper catalyst.The collagen affinity biomatricies prepared by the methods describedabove are in the form of a porous three-dimensional foam scaffold.

Alternatively, the affinity biomatrix may be prepared from hyaluronicacid. For example, the 1, 2 diol in the hyaluronic acid sugar ring isfirst oxidized to an aldehyde, using sodium periodate. Next, an affinitypeptide toward infliximab that has been conjugated to provide a lysineresidue (amine) is reacted with the aldehyde group on the hyaluronicacid to form a Schiff base. The Schiff base is further reacted with areducing agent, such as sodium cyano-borohydride to provide the crudeaffinity biomatrix. The affinity biomatix may be purified while insolution by dialysis, and isolated by lyophylization. The lyophilizedaffinity biomatrix may then be reconstituted, if desired.

The affinity biomatrix is useful as a controlled release device forinfliximab. The controlled release device for infliximab comprises anaffinity biomatrix and infliximab. The amount of infliximab that may beloaded into the affinity biomatrix is dependent upon the amount ofaffinity peptide conjugated to the biocompatible, biodegradable polymer.The amount of affinity peptide present on the biocompatible,biodegradable polymer may be equal to or greater than the desired amountof infliximab in the controlled release device. Therefore there may beat least 1 mole of affinity peptide to 1 mole of infliximab to bedelivered. One of skill in the art will be able to determine how muchinfliximab is needed in the controlled release device for the desiredanti-inflammatory response. The controlled release device may be in theform of sponges, particles, injectable gels or liquids, membranes,films, fibers and fiber based scaffolds, and the like.

In one embodiment, the controlled release device is in the form of aninjectable gel or liquid, where the affinity biomatrix is in an aqueoussolution and contains infliximab. Examples of suitable aqueous solutionsinclude, but are not limited to physiological buffer solution, saline,water, buffered saline, phosphate buffer solution, Hank's balanced saltssolution, PBS, Tris buffered saline, Hepes buffered saline, and mixturesthereof. Polymers useful for preparing an injectable gel or liquiddevice may be selected from the natural or synthetic polymers describedabove. In the case of an injectable liquid or gel, the affinitybiomatrix may be present in the aqueous solution in the amount of fromabout 10 mg/mL to about 150 mg/mL.

The infliximab controlled release device may optionally havedisinfectant and/or antibacterial agents incorporated therein. Suitableagents include, but are not limited to, antimicrobial peptides,quaternary ammonium salts, azide, silver, vancomycin, tobramycin,cefamandol, cephalothin, carbenicillin, amoxicillin, gentamicin, andcombinations thereof. The infliximab controlled release device may alsooptionally have cells incorporated therein. Suitable cell types include,but are not limited to, chondrocytes, osteocytes, osteoblasts, stemcells, pluripotent cells, umbilical cord cells, stromal cells,mesenchymal stem cells, bone marrow cells, embryonic stem cells;precursor cells derived from adipose tissue; peripheral blood progenitorcells; stem cells isolated from adult tissue; genetically transformedcells; and combinations thereof.

The controlled release device for infliximab can be sold as a kitcontaining a sterile affinity biomatrix and sterile lyophilized powderof infliximab. The affinity biomatrix and infliximab contained in thekit can be sterilized using conventional sterilization procedures whichmay include, but are not limited to e-beam, gamma irradiation andaseptic sterile preparation methods.

The infliximab affinity biomatrix may have many applications/indicationswhich include the following. First, the affinity biomatrix may be usedfor the localized, sustained release of infliximab for the treatment ofinflammatory degenerative connective diseases associated with TNF-α,such as osteoarthritis, degenerative intervertebral disc degeneration,and the like. Infliximab will help to suppress the inflammatory reactionand further degeneration. Additionally, the affinity biomatrix can beused for the localized, sustained release of infliximab for thetreatment of inflammatory disorders including plaque psoriasis,rheumatoid arthritis, psoriatic arthritis, adult Crohn's disease,pediatric Crohn's disease, fistulizing Chrohn's disease, ulcerativecolitis, and ankylosing spondylitis. The affinity biomatrix may also beused for the localized, sustained release of infliximab for adhesionprevention, topical applications for the treatment of chronic wounds,and as a tissue repair device.

The controlled release device for inflixmab allows one to locallydeliver infliximab in a controlled manner. The containment of infliximabin the biomatrix will help to increase the half-life of the therapeuticand thus, decrease dosing frequencies and cost.

EXAMPLES Example 1 Infliximab Affinity Peptide Selection using PhageDisplay

Primary peptide phage libraries with high complexity (10⁹ peptidessequences per library) were used to select for affinity peptides towardinfliximab. More specifically, biotinylated-infliximab was immobilizedon streptavidin-coated magnetic beads and phage which display thepeptide sequences on the pIX minor coat protein were introduced. Thephage that did not bind to infliximab were washed away and the phagethat did bind to infliximab were isolated and amplified. The isolatedphage that bound to infliximab were used as input for the second roundof the selection following the procedure stated above for a total ofthree rounds.

Prior to starting the solution based panning, all plasticware wasblocked with 3% milk in tris buffered saline containing Tween 20 (TBST)and allowed to incubate at 4° C. overnight. Streptavidin-coated magneticbeads sold under the tradename DYNABEADS M280 by Invitrogen, GrandIsland, N.Y., (cat #602.10) were washed three times with phosphatebuffered saline (PBS). Next, the beads were loaded with biotinylatedinfliximab at a concentration of 150 micrograms per milliliter.Infliximab was obtained from Janssen Pharmaceuticals, Inc., Radnor, Pa.After loading, the infliximab-coated magnetic beads were washed threetimes with PBD then blocked with 3% milk in TBST for one hour at roomtemperature. Meanwhile, eppendorf tubes were blocked (one per library)using 1 mL of 3% dehydrated milk in TBST. Two MC1061F′ GII E. colicultures were started in 2× YT media supplemented with tetracycline on ashaker at 37° C. Next, the phage libraries were blocked prior to theselection. The blocking solution in the eppendorf tubes was thendiscarded and replaced with 200 microliters of the respective phagelibrary and 800 microliters of 3% milk in TBST. The libraries wereallowed to block with tumbling at room temperature for one hour. Tostart the selection, 1 milliliter of resin was captured using a magnetand 1 milliliter of the respective blocked phage library was introduced.This was allowed to tumble at room temperature for one hour. The volumefor each panning round was kept constant at 1 milliliter. To remove anyloosely bound phage, the phage-bound, infliximab coated magnetic beadswere washed manually with PBS containing 0.05% Tween-20 for a total oftwo washes followed by one wash with PBS. Following the last wash, 600microliters of mid-log phage MC1061F′ GII E. coli were introduced toeach eppendorf tube. The beads were then resuspended using gentleinversions and incubated at 37° C. for 30 minutes. The infected bacteriawere grown for 4 hours at 37° C. in 50 mL of 2× YT media supplementedwith tetracycline. Next, the bacteria were separated by centrifugationand phage were precipitated using a PEG/NaCl solution. ThePEG-precipitated phage were used in round two and the above process wasrepeated for a total of three cycles of the selection process.

At the end of the third round the phage that bound to infliximab weresequenced to determine the peptide which is responsible for binding toinfliximab. The phage-infected bacteria were plated out for singleplaques in top agar with MC1061F′GII E. coli. The resultant plaques wereused to isolate phage that bind to infliximab using ELISA. Once singlephage were identified, the phage that bound to infliximab wereamplified, PEG-precipitated and sequence analysis was performed.Peptides having affinity toward infliximab are listed in FIG. 1.

These phage were used for the phage titration described in Example 2 andthe confirmatory and cross-reactivity phage ELISA assay described inExample 3.

Example 2 Infliximab Selection Output Phage Titration

A phage titration was performed to determine the concentration of phagewhich resulted from the PEG precipitation of the phage that bound toinfliximab described in Example 1.

E. coli MC1061F′GII were grown in 2× YT media supplemented withtetracycline for 2-3 hours at 37° C. on a shaker (180-250 rpm). In a96-well plate, phage dilutions were performed, assuming the PEGprecipitated stock solutions contain 10¹² phage/mL. Briefly, 100microliters of 2× YT media was introduced to the wells of a 96-wellplate. To column 1, 10 microliters of respective phage stock solutionswere introduced. Serial 1:10 dilutions were performed across the plateto column 12 resulting in phage concentrations of 10¹⁰, 10⁹, 10⁸, 10⁷,10⁶, 10⁵, 10⁴, 10³, 10², 10¹, 10⁰, 10⁻¹, respectively, where each rowcontains a distinct phage. For phage plaque growth, 1.5 microliters ofeach respective phage dilution series was introduced to anLB/Tetracycline/X-Gal agar plate coated with a solidified topagar/bacteria suspension. Plates were incubated upside down at 37° C.overnight. After overnight incubation, a dilution was chosen where thephage plaques are well separated and can be counted. The followingformula was used to calculate the titer: (#plaques)(66.7)(10^(dilution number−1))×100=phage/mL, where 66.7 is thedilution factor of the phage in each well and the dilution number is thenumber of dilutions performed until phage plaques could be counted.Multiplication by 100 yields the number of phage/mL in the stock phagesuspension.

Calculated phage numbers are shown in Table 1. Phage concentrationsobtained were used to perform the dilutions for the confirmatory andcross-reactivity phage ELISA assay described in Example 3.

TABLE 1 SEQ ID NO. Phage/mL 1 3.3 × 10¹³ 2 3.3 × 10¹³ 3 4.0 × 10¹² 4 1.4× 10¹² 5 1.4 × 10¹² 6 1.4 × 10¹² 7 1.4 × 10¹² 8 1.4 × 10¹² 9 1.4 × 10¹²10 1.4 × 10¹² 11 1.4 × 10¹² 12 1.4 × 10¹² 13 1.2 × 10¹² 14 1.0 × 10¹² 151.2 × 10¹² 16 1.0 × 10¹² 17 1.2 × 10¹² 18 1.2 × 10¹² 19 1.3 × 10¹¹ 206.0 × 10¹² 21 6.0 × 10¹² 22 NA (Did not titer) 23 5.3 × 10¹³

Example 3 Confirmatory and Cross-Reactivity Phage Enzyme LinkedImmunosorbent Assay (ELISA)

In this experiment, we are confirming the binding of the phageidentified in Example 1 to infliximab. In addition, we are also testingthat the phage do not exhibit significant binding to monoclonal antibody(mAb) competitor, human IgG, human serum albumin (HSA) and collagen.

96-well black ELISA plates were coated with streptavidin (Columns 1through 4), mAb competitor (Columns 5 and 6), hIgG (Columns 7 and 8),human serum albumin (Columns 9 and 10) and collagen (Columns 11 and 12),at a concentration of 5 micrograms/mL in PBS. Plates were incubatedovernight at 4° C. Plates were washed 3 times with TBST using a platewasher. To the streptavidin-coated wells in columns 3 and 4, 100microliters of a 5 micrograms/mL solution of biotinylated-infliximab wasintroduced and allowed to sit at room temperature for 30 minutes. To allother wells, 100 microliters of PBS was introduced. The plates wereagain washed 3 times with TBST using the plate washer. The wells werethen blocked with 250 microliters of a 3% dehydrated milk suspension inTBST and allowed to sit at room temperature for a minimum of 1 hour.Using phage concentrations determined by the phage titrations, dilutionsof the phage stock solutions were performed in a separate 96-well plateto result in phage concentrations of 10¹⁰, 10⁹, 10⁸, 10⁷, 10⁶ and 10⁵phage/well. After blocking, the ELISA plate was washed 3 times with TBSTand 100 microliters of the phage dilutions were introduced to therespective ELISA plates and allowed to sit at room temperature for aminimum of 1 hour. The ELISA plate was again washed 3 times with TBSTusing the plate washer and 100 microliters of an horseradish peroxidase(HRP)-conjugated Anti-M13 Phage Mab (1:5000 dilution in PBS) wasintroduced to each well. The plates were allowed to sit at roomtemperature for 1 hour after which time the plates were washed 3 timeswith TBST. Next, a peroxidase-based (POD) HRP-substrate was introducedto each well. The luminescence was read using a luminometer set with again of 150.

The phage ELISA data in FIG. 3 and FIG. 4 show relative luminescence andhave an N value of 2. Data in FIG. 3 confirms the binding of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ IDNO: 21, SEQ ID NO: 22, SEQ ID NO: 23 to infliximab. Data in FIG. 4A,FIG. 4B, FIG. 4C and FIG. 4D show that the phage are minimallycross-reactive with mAb competitor, hIgG, HSA and collagen,respectively.

Example 4 Infliximab Affinity Peptide ELISA Assay

Concentration dependent binding efficiency of infliximab to the affinitypeptides was evaluated using ELISA.

96-well black ELISA plates were coated with goat anti-human Fc (JacksonImmunoresearch Laboratories, Inc., West Grove, Pa., cat. #109-006-098)at a concentration of 5 micrograms/mL in PBS. Plates were incubatedovernight at 4° C. Plates were washed 3 times with TBST using a platewasher. To the anti-Fc-coated wells, 100 microliters of a 20 microgramsper milliliter solution of infliximab was introduced and allowed to sitat room temperature for one hour. To all other wells, 100 microliters ofPBS was introduced. The plates were again washed 3 times with TBST usingthe plate washer. The wells were then blocked with 250 microliters of a3% dehydrated milk suspension in TBST and allowed to sit at roomtemperature for a minimum of 1 hour. Biotinylated, chemicallysynthesized peptides having SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 10,and SEQ ID NO: 13 were prepared using standard, automatedFluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesisprocedures. Each peptide solution for the assay was prepared from a 500micromolar stock solution. The stock solution was serially diluted inassay buffer resulting in peptide concentrations of 500, 166.6, 55.5,18.5, 6.2, 2.1, 0.69, 0.23, 0.076, 0.025, 0.0085 and 0.0028 micromolar.

After blocking, the ELISA plate was washed 3 times with TBST and 100microliters of the serially diluted peptides were added to thecorresponding wells of the ELISA plate and allowed to sit at roomtemperature for a minimum of 1 hour. The ELISA plate was again washed 3times with TBST using the plate washer and 100 microliters ofHRP-conjugated streptavidin (1:10000 dilution in PBS) was introduced toeach well. The plates were allowed to sit at room temperature for 1 hourafter which time the plates were washed 3 times with TBST. Next, a PODHRP-substrate was introduced to each well. The luminescence was readusing a luminometer set with a gain of 150.

The ELISA data in FIG. 5 was normalized to molar concentration based onthe molecular weights of the peptide sequences. The data in FIG. 5 showsthe concentration-dependent binding of the peptides to infliximab.Peptide sequences were ranked according to their concentration-dependentbinding to infliximab as follows; SEQ ID NO: 2>SEQ ID NO: 10>SEQ ID NO:3>SEQ ID NO: 13.

Example 5 Surface Plasmon Resonance

Peptide binding affinity to infliximab was evaluated for SEQ ID NO: 2and SEQ ID NO: 10 by surface plasmon resonance measurements.Measurements were performed on a surface plasmon resonance-basedbiosensor sold under the tradename BIACORE S51 (Biacore Life Sciences,Piscataway, N.J.) using a streptavidin Series-S sensor chip.Biotinylated peptides were immobilized on the sensor chip at aconcentration of 0.1 and 0.2 micrograms/mL (For SEQ ID NO: 8 and SEQ IDNO: 10, c=1 and 10 micrograms/mL). Infliximab was flowed over theaffinity peptide functionalized chip at concentrations of 0, 0.78, 1.56,3.125, 6.25, 12.5, 25, 50 and 100 nM and changes in the refractive indexwere recorded. Data were fit to a 1:1 Langmuir binding model usinganalysis software sold under the tradename BIACORE by Biacore LifeSciences, Piscataway, N.J.

The data shown in Table 1 are average equilibrium dissociation constantswith an N value of 2. Data show average equilibrium dissociationconstants between 9 and 18 nM.

TABLE 1 K_(D) (M) SEQ ID: 2 (1011A) 17.1 × 10⁻⁹ SEQ ID: 10 (1067B) 9.17× 10⁻⁹

Example 6 Preparation of Collagen Biomatrix

A 40 mg/mL solution of 80% fibrous Type I collagen (32 mg) and 20%soluble Type I collagen (8 mg) in water was stirred overnight at 4° C.This suspension was then homogenized in a blender for 3 cycles (30seconds to 1 min/cycle). Approximately 5 mL of the suspension wastransferred to a mold with the dimensions of 5 cm×5 cm and a height of0.5 cm. The collagen mixture was leveled with a straight edge spatula toensure an even height distribution of the collagen. Next, the sample wasdegassed then lyophilized using the cycle shown in Table 3. The sampleswere then thermally dehydrated using a temperature controlled vacuumchamber. The foam collagen biomatrix was then cut into 6 mm discs usinga biopsy punch and stored at room temperature under dry nitrogen purge.The biomatrices prepared in this example were used for functionalizationas described in Example 7.

TABLE 3 Temperature (° C.) Time (minutes) Pressure (mTorr) −40 60 500−25 300 100 −20 600 50 −10 300 50 −5 180 50 0 120 50 10 120 50 20 120 50

Example 7 Preparation of the Azide-Modified Collagen Biomatrix

To prepare the azide-modified collagen biomatrix, a 6 mm collagen spongedisc, prepared as described in Example 6, was placed in 1 mL of 50millimolar borate buffer in an eppendorf tube and allowed to swell for 1hour at room temperature. The sponge was removed from the above bufferand placed in 1.5 mL of fresh 50 millimolar borate buffer containing 5millimolar of the NHS-PEG4-Azide (Thermo Fisher Scientific, Waltham,Mass., cat #26130). The reaction mixture was allowed to tumble at roomtemperature overnight (approximately 16-18 hours). The sponge was thenremoved from the reaction mixture and gently squeezed to remove excessfluid. The sponge was then washed using 10 milliliters of PBS whiletumbling at room temperature for two hours. The washing process wasrepeated for a total of three washes. The azide-modified biomatricesdescribed in this example were used to conjugate the affinity peptidesas described in Example 8.

Example 8 Conjugation of Infliximab Affinity Peptide to Azide-ModifiedCollagen Biomatrix

For peptide conjugation, a 6 mm collagen sponge disc, prepared asdescribed in Example 7, was placed in 1.8 mL of 20% DMSO/H₂O in aneppendorf tube and tumbled for 30 min. at room temperature. The spongewas removed from the above buffer and placed in 1.8 mL of fresh 20%DMSO/H₂O containing 1 milliolar affinity conjugation peptide. Theaffinity biomatricies were prepared with affinity conjugation peptide ofSEQ ID NO: 2 and SEQ ID NO: 10 (sequences SEQ ID NO: 24 and SEQ ID NO:25 shown in FIG. 6A), respectively. Peptides were prepared usingstandard, automated Fluoroenylmethyloxycarbonyl (Fmoc) solid phasepeptide synthesis procedures. The sample was tumbled at room temperaturefor 1 hour. Next, 36 microliters of a 10 millimolar copper acetate 50millimolar ascorbic acid solution in water was introduced to eacheppendorf containing a collagen sponge. This was then tumbled for 2 daysat room temperature. Next, the sponge was rinsed by tumbling in 10 mL ofPBS for 2 hours. This was repeated for a total of three rinse cycles.Lastly, the sponge was removed from the PBS and allowed to dry on thelyopholizer overnight. Three control samples were also used in the aboveprocedure. 1=Collagen Sponge+Buffer, 2=Collagen Sponge+Peptide/Buffer,and 3=Collagen Sponge+Copper Acetate/Ascorbic Acid. For all samples, thesupernatants were lyophilized to a dry powder and analyzed usingRP-Analytical HPLC. Data show approximately 10-30% conjugationefficiency was achieved.

Example 9 Conjugation of Infliximab Affinity Peptide to HA Biomatrix

A hyaluronic acid (HA) hydrogel solution (20 mg/mL in PBS), was dilutedwith sterile filtered water to a final concentration of 2 mg/mL. Thissolution was allowed to stir at room temperature for 15 minutes. For theactivation of HA, a 100 mM sodium periodate solution in water wasintroduced to the diluted HA solution to a final concentration of 10 mM.The solution was then covered with aluminum foil and allowed to stir atroom temperature for 30 minutes. Next, a 1M solution of D-Mannitol inwater was introduced to a final concentration of 0.1 M and was allowedto stir at room temperature for 10 minutes. The activated HA was thendialyzed in water using a 3.5K dialysis cassette. The water was changedevery two hours for a total of two cycles and then dialysis wasperformed overnight at room temperature. After dialysis, the water wasremoved from the sample by lyopholization. For the conjugation of theaffinity peptide (sequences shown in FIG. 6B) to the activated HA, 20 mgof activated HA was introduced to 5 mL of sterile filtered waterresulting in a 4 mg/mL activated HA solution. In a separate vial, 5 mgof affinity peptide (SEQ ID NO: 26 or SEQ ID NO: 27) was dissolved in 5mL of 20 mM sodium phosphate buffer (pH 7.5) resulting in a 1 mg/mLpeptide solution. Next, a 1:1 dilution of activated HA and peptide wasperformed (total volume=10 mL) resulting in a 2 mg/mL activated HA:0.5mg/mL peptide solution. This was allowed to stir at room temperature forfour hours. Next, sodium cyanoborohydride was introduced at a finalconcentration of 5 mM and allowed to stir at room temperature for 30minutes. The solution was then dialyzed in water and lyophilized toobtain the purified affinity biomatrix.

Example 10 In vitro Bioactivity of Infliximab in the Presence of theAffinity Peptides

In this experiment, we determined if the affinity peptides wouldinterfere with the binding of tumor necrosis factor alpha (TNF-α) usingWEHI-13VAR mouse fibroblasts. The cell viability was monitored thepresence of infliximab with and without affinity peptides using the3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay.

WEHI-13VAR cells (mouse fibroblast cell line) were plated out in wellsof a 96 well plate two hours prior to running the experiment at 50000cells per well. A stock solution of 20 nanograms per milliliterrecombinant human TNF-α (R&D Systems, 210-TA/CF) was prepared in culturemedia containing 7.5 micrograms per milliliter of actinomycin D. Serialdilutions of infliximab were prepared in a concentration range of 0-66nanomolar in the presence or absence of peptides SEQ ID NO: 2, SEQ IDNO: 10 and SEQ ID NO: 13 at a constant concentration of 66 nanomolarresulting in peptide:infliximab ratios of 1, 4, 16, 64, 256, 1024, 4094and 16384. 160 microliters of the respective infliximab:peptide solutionand 40 microliters of the TNF-α solution were preincubated for 30minutes prior to the addition to the wells containing the WEHI-13VARcells. Next, cells were stimulated with 50 microliters of theTNF-α:infliximab:peptide solution and allowed to incubate overnight in ahumidified incubator at 37 degrees Celcius with 5% CO₂. After overnightincubation, cell viability was assessed using an MTT cell viabilityassay (R&D Systems, Inc., Minneapolis, Minn., cat #4890-050-K) followingthe product insert.

FIG. 7 shows that SEQ ID NO: 2, SEQ ID NO: 10 and SEQ ID NO: 13 haveminimal effect on the viability of WEHI-13VAR mouse fibroblasts in thepresence of infliximab. This suggests that SEQ ID NO: 2, SEQ ID NO: 10and SEQ ID NO: 13 do not interfere with the bioactivity of infliximab.

Example 11 In vitro Release of Infliximab from Collagen Discs

In this experiment, we have evaluated the in vitro controlled release ofinfliximab from the affinity biomatrix under simulated physiologicalconditions. For the release studies, 6 mm diameter discs of affinitybiomatrix as prepared by the methods described in Example 6, Example 7,and Example 8, were placed in a well of a 24 well cell culture plate.The sponges were loaded with either 50 micrograms or 200 micrograms ofinfliximab and allowed to sit at room temperature for 60 minutes. A 6 mmdiameter disc of the affinity peptide-free collagen as prepared inExample 6 was used as a control. The loaded collagen sponges were thentransferred to a well of a 24 well cell culture plate containing 1.5 mLof PBS supplemented with heat denatured fetal bovine serum (FBS) at aconcentration of 1 mg/mL. At the respective time points, the media wasremoved and transferred to an eppendorf tube then stored at 4° C. untilanalysis. The wells containing the collagen discs were replenished with1.5 mL of fresh 2% FBS in PBS. The release media was analyzed forinfliximab content using ELISA.

Data in FIG. 8A shows the affinity biomatrix controlled release deviceloaded with 50 micrograms of infliximab has a cumulative release ofinfliximab of approximately 40% (SEQ ID NO: 24) and 55% (SEQ ID NO: 25)by weight at the 28 day time point whereas the unmodified controlcollagen sponge releases approximately 100% by weight. Data in FIG. 8Bshows the affinity biomatrix controlled release device loaded with 200micrograms of infliximab has a cumulative release of infliximab ofapproximately 89% (SEQ ID NO: 24) and 80% (SEQ ID NO: 25) by weight atthe 28 day time point whereas the unmodified control collagen spongereleases approximately 100% by weight. The initial burst release ofinfliximab from the affinity peptide biomatrix controlled release deviceis significantly reduced as compared to the unmodified control collagensponge.

Example 12 Mutant Phage ELISA

Site-directed mutagenesis was performed on affinity peptide SEQ ID NO:18 to obtain a linear version of the peptide and to understand whichamino acids are important for binding to infliximab. Mutants of theabovementioned affinity peptide were constructed by randomizing residues3 through 6 and residues 15 through 18 of the wild type affinity peptideSEQ ID NO: 18, AG-XXXX-PWPPTAES-XXXX, where X denotes any of the 20natural amino acids (SEQ ID NO: 32).

The respective custom oligonucleotide was obtained from Integrated DNATechnologies, Inc. (Coralville, Iowa). The oligonucletides were insertedinto the pIX gene using standard protocols. First, the oligonucletideswere phosphorylated using T4 polynucleotide kinase. Next, thephosphorylated oligonucletides were annealed to the ssDNA template.Using the Kunkel method, the annealed DNA was ligated using T4 DNAligase and T7 DNA polymerase. The resultant DNA was isolated usingisopropanol precipitation and a gel was run to confirm the reaction.Lastly, electro-competent E. coli cells were transformed with thepurified DNA, plated out onto 2× YT/Tet/Xgal agar plates and wereallowed to grow overnight to isolate single colonies. An aliquot of thetransformed cells was allowed to grow overnight at 37° C. while shakingin 50 mL 2× YT/tet media. The phage was PEG precipitated following theprocedure in Example 1. A phage titration was performed to determine theconcentration of phage for the ELISA assay as described in Example 2.ELISA assays were performed following the procedure in Example 3 toidentify fragments of SEQ ID NO: 18 that bind to infliximab. Data inFIG. 9 show the mutant affinity peptides, whose sequences are listed inFIG. 10, are binding to infliximab.

We claim:
 1. An affinity peptide toward infliximab having the peptidesequence of SEQ ID NO: 2 or SEQ ID NO:
 10. 2. A controlled releasedevice comprising an affinity biomatrix, further comprising a polymerand an affinity peptide toward infliximab having the peptide sequence ofSEQ ID NO: 2 or SEQ ID NO: 10, and infliximab.
 3. The controlled releasedevice of claim 2, wherein the polymer is a natural polymer.
 4. Thecontrolled release device of claim 3, wherein the natural polymer isselected from the group consisting of collagen, elastin, keratin, silk,polysaccharides, GAGs, and combinations thereof.
 5. The controlledrelease device of claim 4 wherein the polysaccharide is selected fromthe group consisting of starch, pectin, cellulose, alkyl cellulose,alkylhydroxyalkyl cellulose, hydroxyalkyl cellulose, cellulose sulfate,salts of carboxymethyl cellulose, carboxymethyl cellulose, carboxyethylcellulose, chitin, carboxymethyl chitin, hyaluronic acid, salts ofhyaluronic acid, alginate, cross-linked alginate/alginic acid, propyleneglycol alginate, glycogen, dextran, dextran sulfate, curdlan, pectin,pullulan, xanthan, chondroitin, chondroitin sulfates, carboxymethyldextran, carboxymethyl chitosan, chitosan, heparin, heparin sulfate,heparan, heparan sulfate, dermatan sulfate, keratan sulfate,carrageenans, chitosan, starch, amylose, amylopectin,poly-N-glucosamine, polymannuronic acid, polyglucuronic acid, andpolyglucuronic acid.
 6. The controlled release device of claim 3,wherein the natural polymer is collagen.
 7. The controlled releasedevice of claim 3, wherein the natural polymer is decellularized tissueselected from the group consisting of skin, periosteum, perichondrium,synovium, fascia, mesenter, bone and sinew.
 8. The controlled releasedevice of claim 6, wherein the collagen is in the form of sponges,particles, injectable gels, injectable liquids, membranes, films, fibersand fiber based scaffolds.
 9. A kit comprising an affinity biomatrix,further comprising a polymer and an affinity peptide toward infliximabhaving the peptide sequence of SEQ ID NO: 2 or SEQ ID NO: 10, andinfliximab.